RESUMEN
A series of imidazolinylindole derivatives were discovered as novel kallikrein 7 (KLK7, stratum corneum chymotryptic enzyme) inhibitors. Structure-activity relationship (SAR) studies led to the identification of potent human KLK7 inhibitors. By further modification of the benzenesulfonyl moiety to overcome species differences in inhibitory activity, potent inhibitors against both human and mouse KLK7 were identified. Furthermore, the complex structure of 25 with mouse KLK7 could explain the SAR and the cause of the species differences in inhibitory activity.
Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Imidazolinas/farmacología , Indoles/farmacología , Calicreínas/antagonistas & inhibidores , Animales , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Imidazolinas/síntesis química , Imidazolinas/química , Indoles/síntesis química , Indoles/química , Calicreínas/metabolismo , Ratones , Estructura Molecular , Relación Estructura-ActividadRESUMEN
A series of 1,3,6-trisubstituted 1,4-diazepan-7-ones were prepared as kallikrein 7 (KLK7, stratum corneum chymotryptic enzyme) inhibitors. Previously reported compounds 1-3 were potent human KLK7 inhibitors; however, they did not exhibit inhibitory activity against mouse KLK7. Comparison of the human and mouse KLK7 structures reveals the cause of this species differences; therefore, compounds that could inhibit both KLK7s were designed, synthesized, and evaluated. Through this structure-based drug design, compound 22g was identified as an inhibitor against human and mouse KLK7, and only one of the enantiomers, (-)-22g, exhibited potent inhibitory activity. Furthermore, the crystal structure of mouse KLK7 complexed with 22g enabled the elucidation of structure-activity relationships and justified 22g as a valuable compound to overcome the species differences.
Asunto(s)
Azepinas/química , Calicreínas/metabolismo , Inhibidores de Proteasas/síntesis química , Secuencia de Aminoácidos , Animales , Azepinas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Calicreínas/antagonistas & inhibidores , Ratones , Inhibidores de Proteasas/metabolismo , Estructura Terciaria de Proteína , Alineación de Secuencia , Especificidad de la Especie , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
A novel series of 1,3,6-trisubstituted 1,4-diazepan-7-ones were investigated as human kallikrein 7 (KLK7, stratum corneum chymotryptic enzyme) inhibitors. Based on the X-ray co-crystal structure of compound 1 bound to human KLK7, the derivatives of this scaffold were designed, synthesized, and evaluated. Through structure-activity relationship studies focused on the side chain located in the prime site region of the enzyme, representative compounds 15, 33a, and 35a were identified as highly potent and selective inhibitors of human KLK7.
Asunto(s)
Azepinas/farmacología , Calicreínas/antagonistas & inhibidores , Azepinas/síntesis química , Azepinas/química , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Calicreínas/química , Simulación del Acoplamiento Molecular , Estructura Molecular , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Compound 1, composed of a 1,3,6-trisubstituted 1,4-diazepane-7-one, was discovered as a novel human kallikrein 7 (KLK7, stratum corneum chymotryptic enzyme, SCCE) inhibitor, and its derivatives were synthesized and evaluated. Structure-activity relationship studies of the amidoxime unit and benzoic acid part of this new scaffold led to the identification of 25 and 34, which were more potent than the hit compound, 1. The X-ray co-crystal structure of compound 25 and human KLK7 revealed the characteristic interactions and enabled explanations of the structure-activity relationship.
Asunto(s)
Azepinas/farmacología , Descubrimiento de Drogas , Calicreínas/antagonistas & inhibidores , Inhibidores de Serina Proteinasa/farmacología , Azepinas/síntesis química , Azepinas/química , Relación Dosis-Respuesta a Droga , Humanos , Calicreínas/metabolismo , Estructura Molecular , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/química , Relación Estructura-ActividadRESUMEN
Excessive proliferation of epidermal keratinocytes is a typical aspect of chronic skin diseases such as psoriasis. In the present study, the effect of phosphodiesterase 7A (PDE7A) inhibitor ASB16165 on proliferation of keratinocytes was investigated to examine the role of PDE7A in keratinocyte proliferation and the possible therapeutic relevance of PDE7A inhibition in psoriasis. Topical application of ASB16165 inhibited the increase of thickness of skin as well as epidermis in a skin inflammation model induced by repeated painting of 12-O-tetradecanoylphorbol-13-acetate (TPA) in a concentration-dependent manner. The ASB16165 treatment also suppressed the increase in the number of Ki67-positive keratinocytes in the model, showing the disturbance of keratinocyte proliferation by the treatment. In addition, both ASB16165 and dibutyryl cAMP significantly decreased the proliferation of human keratinocytes in vitro, suggesting that PDE7A participates in keratinocyte proliferation probably by controlling intracellular cAMP, while the contribution of other mechanism(s) is not completely denied. The findings in the present study indicate that the effect of ASB16165 on skin and epidermal hyperplasia in the TPA-induced skin inflammation is mediated, at least in part, by the inhibition of keratinocyte proliferation. The inhibitors for PDE7A including ASB16165 might be useful for the treatment of psoriasis.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 7/antagonistas & inhibidores , Epidermis/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Inhibidores de Fosfodiesterasa/farmacología , Psoriasis/tratamiento farmacológico , Pirazoles/farmacología , Piel/efectos de los fármacos , Tiofenos/farmacología , Administración Cutánea , Animales , Bucladesina/farmacología , Células Cultivadas , Modelos Animales de Enfermedad , Epidermis/patología , Femenino , Humanos , Queratinocitos/patología , Ratones , Ratones Endogámicos BALB C , Inhibidores de Fosfodiesterasa/administración & dosificación , Psoriasis/inducido químicamente , Pirazoles/administración & dosificación , Piel/patología , Acetato de Tetradecanoilforbol , Tiofenos/administración & dosificaciónRESUMEN
An intravenous injection of Concanavalin A (Con A) elevated the serum level of alanine aminotransferase (ALT) activity, a marker for liver damage, and an oral administration of PDE7A inhibitor SUN11817 suppressed the increase of ALT activity in a dose-dependent manner. Histological analysis revealed that Con A injection caused extensive liver damage, and that the SUN11817 treatment improved the degenerative change in the liver. In addition, SUN11817 inhibited not only the production of IL-4 and TNF-alpha in the Con A-induced hepatitis model but also that in vitro by murine splenocytes stimulated with alpha-galactosylceramide, an activator specific for NKT cells. The Con A injection to mice also induced expression of Fas ligand (FasL) on NKT cells, which was significantly prevented by SUN11817. As NKT cells are known to contribute to the pathogenesis in Con A-induced hepatitis by producing cytokines such as IL-4 and TNF-alpha and inducing FasL-mediated hepatocyte injury, it is thought that PDE7A inhibitor SUN11817 improves liver injury in the Con A model by blocking cytokine production and FasL expression in NKT cells. PDE7A might be a novel pharmaceutical target for hepatitis.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Concanavalina A , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 7/antagonistas & inhibidores , Células T Asesinas Naturales/efectos de los fármacos , Pirazoles/uso terapéutico , Tiofenos/uso terapéutico , Alanina Transaminasa/sangre , Animales , Recuento de Células , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Proteína Ligando Fas/metabolismo , Femenino , Galactosilceramidas/farmacología , Interleucina-4/sangre , Interleucina-4/metabolismo , Hígado/citología , Hígado/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Células T Asesinas Naturales/metabolismo , Pirazoles/farmacología , Bazo/efectos de los fármacos , Bazo/metabolismo , Tiofenos/farmacología , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
A possible involvement of phosphodiesterase 7A (PDE7A) in proliferation and function of NKT cells was examined using ASB16165, a selective inhibitor for PDE7A. Stimulation of isolated murine NKT cells with anti-CD3 antibody plus IL-2 induced not only cell proliferation but production of cytokines including IFN-gamma, TNF-alpha, IL-17 and IL-22. ASB16165 significantly inhibited the CD3/IL-2-stimulated cell proliferation and production of all the cytokines examined. Forskolin (an activator of adenylyl cyclase) and dibutyryl cAMP also exerted inhibitory effects on the cell proliferation and cytokine production of NKT cells. In addition, Rp-8-Br-cAMPS, an inhibitor of protein kinase A (PKA), reversed the suppressive effects of ASB16165 against NKT cells. These results suggest that PKA/cAMP as well as PDE7A is involved in regulation of cell proliferation and cytokine production of NKT cells, and that the inhibitory effects of ASB16165 in NKT cells shown here are mediated by increase in cellular cAMP level. Our findings also raise the possibility that PDE7A inhibitor including ASB16165 may be useful for treatment of the diseases in which NKT cells have pathogenic roles.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 7/metabolismo , Citocinas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Células T Asesinas Naturales/efectos de los fármacos , Pirazoles/farmacología , Tiofenos/farmacología , Animales , Proliferación Celular , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 7/antagonistas & inhibidores , Citocinas/biosíntesis , Femenino , Ratones , Ratones Endogámicos C57BL , Células T Asesinas Naturales/inmunologíaRESUMEN
Possible role of phosphodiesterase 7A (PDE7A) in skin inflammation was examined using ASB16165, a specific inhibitor for PDE7A. Epicutaneous application of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse ear resulted in induction of skin edema, and topical treatment with ASB16165 inhibited the induction of skin edema in a dose-dependent manner. The TPA challenge also increased the level of TNF-alpha at the application site, and the ASB16165 treatment reduced the TNF-alpha level in the skin. In addition, ASB16165 suppressed the production of TNF-alpha by human keratinocytes stimulated in vitro with TPA and calcium ionophore. Forskolin, an activator of adenylyl cyclase, as well as dibutyryl cAMP also showed inhibitory effect on the TNF-alpha production in the cells, suggesting involvement of cAMP in TNF-alpha generation. These results demonstrate that PDE7A might regulate TNF-alpha production in keratinocytes in a cAMP-dependent fashion. As immunostaining analysis revealed that PDE7A is expressed in the epidermis and TNF-alpha is known to contribute to the TPA-induced edema, it is possible that the inhibitory effect of ASB16165 on skin edema in mouse TPA-induced dermatitis model is mediated by suppression of TNF-alpha production. This is the first report suggesting the association of PDE7A with the function of keratinocytes. ASB16165 will be useful as an agent for skin inflammation in which TNF-alpha plays a pathogenic role (e.g. psoriasis).
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 7/antagonistas & inhibidores , Edema/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Pirazoles/farmacología , Piel/efectos de los fármacos , Acetato de Tetradecanoilforbol/toxicidad , Tiofenos/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Edema/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Epidermis/efectos de los fármacos , Epidermis/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Inflamación/patología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Ratones , Ratones Endogámicos BALB C , Pirazoles/uso terapéutico , Piel/metabolismo , Piel/patología , Tiofenos/uso terapéutico , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Phosphodiesterase 7A (PDE7A) has been suggested to be involved in activation of T lymphocytes. In the present study, a possible involvement of PDE7A in function of preactivated T cells (i.e. T lymphoblasts) was investigated using ASB16165, an inhibitor for PDE7A. ASB16165, which has an IC50 value of 15 nM for human PDE7A, suppressed IL-12-induced IFN-gamma production by T lymphoblasts which have been prepared by stimulating mouse T cells with anti-CD3 antibody. In the same experiment, rolipram, a PDE4-specific inhibitor, showed similar effect, while calcineurin antagonist FK506 did not. Forskolin (an adenylyl cyclase activator) and dibutyryl cAMP also inhibited the IL-12-induced IFN-gamma synthesis. Rp-8-Br-cAMPS, an inhibitor of protein kinase A (PKA), reduced the suppressive effect of ASB16165 on the IFN-gamma production by T lymphoblasts. The rescue of IFN-gamma production by Rp-8-Br-cAMPS was also observed in the inhibition by rolipram and forskolin. These findings suggest that PDE7A may regulate function of activated T cells in a cAMP/PKA-dependent manner, and that PDE4 might share the role. The data in our study also indicate that PDE7 inhibitors such as ASB16165 will be beneficial for the patients with immunological disorders.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 7/antagonistas & inhibidores , Interferón gamma/metabolismo , Interleucina-12/metabolismo , Activación de Linfocitos/efectos de los fármacos , Pirazoles/farmacología , Linfocitos T/efectos de los fármacos , Tiofenos/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Anticuerpos Monoclonales , Complejo CD3 , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Interleucina-12/genética , Interleucina-12/inmunología , Ratones , Ratones Endogámicos C57BL , Inhibidores de Fosfodiesterasa 4 , Rolipram/farmacología , Linfocitos T/enzimología , Linfocitos T/inmunología , Tionucleótidos/farmacologíaRESUMEN
In the present study, possible role of phosphodiesterase 7 (PDE7) in development and function of cytotoxic T lymphocyte (CTL) was examined using ASB16165, a specific inhibitor for PDE7. ASB16165 inhibited generation of CTL activity in mixed lymphocyte reaction (MLR), in which splenocytes from C57BL/6N mice were stimulated with those from BALB/c mice. Flow cytometric analysis revealed that ASB16165 suppressed induction of activated CD4+ as well as CD8+ T cells in MLR. In cell division analyses using 5-carboxyfluorescein diacetate succinimide ester (CFSE), ASB16165 was shown to markedly inhibit proliferation of CD4+ and CD8+ T cells. In addition, ASB16165 reduced effector function of CTL, while the effect was less than that observed in CTL induction in MLR. Forskolin and dibutyryl cAMP also inhibited both the induction and effector function of CTL. PDE4 inhibitor rolipram showed similar but weaker inhibition for the development and proliferation of CD8+ T cells compared with ASB16165, and failed to impair effector function of CTL. These findings suggest that PDE7 but not PDE4 has the major role in induction and function of CTL in mice, and that the effect might be mediated by elevation of intracellular cAMP level. ASB16165 may be useful for treatment of the diseases in which CTL has a pathogenic role (e.g. autoimmune diseases).
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 7/antagonistas & inhibidores , Inhibidores de Fosfodiesterasa/farmacología , Pirazoles/farmacología , Linfocitos T Citotóxicos/efectos de los fármacos , Tiofenos/farmacología , Animales , Bucladesina/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Colforsina/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 7/metabolismo , Femenino , Citometría de Flujo , Inmunosupresores/farmacología , Indicadores y Reactivos , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Rolipram/farmacología , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunología , Tacrolimus/farmacologíaRESUMEN
Serine-O-carbonate derivatives, including peptides having a serine-O-carbonate residue at the N-terminal position, are catalytically transformed into S-substituted cysteine derivatives employing the pyridoxal model having an ionophore function in the presence of Li+; this is the first artificial model mimicking cystathionine Beta-synthase.